Analysis of Cytochrome Oxidase: A New Cell and Molecular Biology Lab

Aerobic organisms use O2 as a final electron acceptor in cellular respiration. High-energy electrons are passed through an electron transport chain that creates a gradient resulting in the production of ATP a process called oxidative phosphorylation. This process takes place within the mitochondrial membrane. An enzyme anchored in the membrane (cytochrome c oxidase) catalyzes the reduction of O2 to water and also transfers protons across the membrane to form the chemiosmotic gradient responsible for oxidative phosphorylation. The general goal of this research was to refine and develop a new laboratory exercise for the Cellular and Molecular Biology class. We isolated cauliflower mitochondria using differential centrifugation and sucrose gradient velocity sedimentation. Mitochondrial fractions were determined using the marker enzyme succinate dehydrogenase which is also located within the mitochondrial membrane. SDS-PAGE analysis of proteins was used in an attempt to determine the purity of cytochrome c oxidase. The mitochondrial DNA was isolated and transferred to a nitrocellulose membrane. Using Southern analysis, an attempt to determine the number of copies of the gene for cytochrome c oxidase present was made.